Interaction Kinetics of Individual mRNA-Containing Lipid Nanoparticles with an Endosomal Membrane Mimic: Dependence on pH, Protein Corona Formation, and Lipoprotein Depletion

Lipid nanoparticles (LNPs) have emerged as potent carriers for mRNA delivery, but several challenges remain before this approach can offer broad clinical translation of mRNA therapeutics. To improve their efficacy, a better understanding is required regarding how LNPs are trapped and processed at the anionic endosomal membrane prior to mRNA release. We used surface-sensitive fluorescence microscopy with single LNP resolution to investigate the pH dependency of the binding kinetics of ionizable lipid-containing LNPs to a supported endosomal model membrane. A sharp increase of LNP binding was observed when the pH was lowered from 6 to 5, accompanied by stepwise large-scale LNP disintegration. For LNPs preincubated in serum, protein corona formation shifted the onset of LNP binding and subsequent disintegration to lower pH, an effect that was less pronounced for lipoprotein-depleted serum. The LNP binding to the endosomal membrane mimic was observed to eventually become severely limited by suppression of the driving force for the formation of multivalent bonds during LNP attachment or, more specifically, by charge neutralization of anionic lipids in the model membrane due to their association with cationic lipids from earlier attached LNPs upon their disintegration. Cell uptake experiments demonstrated marginal differences in LNP uptake in untreated and lipoprotein-depleted serum, whereas lipoprotein-depleted serum increased mRNA-controlled protein (eGFP) production substantially. This complies with model membrane data and suggests that protein corona formation on the surface of the LNPs influences the nature of the interaction between LNPs and endosomal membranes.


FRAP assessment at various pH levels
. FRAP analysis performed on the ncSLB at the various pH employed in the assay. The data showed a stable diffusivity throughout the experiment with a negligible increase in the immobile fraction of the lipids.

LNP preparation and characterization
The structures of the lipids used for the LNP formation (see Materials and Methods in Main text) are shown in Figure S2A together with size and concentration determination of the LNPs after 50 times dilution in PBS using DLS measurements (ZetaSizer Nano ZS from Malvern Instruments Ltd) to be 78 nm and ~1.3×10 13 particle/ml), respectively ( Figure S2B). The volume-weighted particle size distributions were calculated using a particle refractive index of 1.45. The encapsulation and concentration of mRNA were determined using the RiboGreen assay.
The number of MC3 lipids per LNPs was determined from the final 3.08 molar ratio of the negative to positive charge in the formulation solution, resulting in an effective molar ratio of 3.17 considering an encapsulation efficiency of 97%. This results in 0.134 mg/mL / 363 g/mol 3.17 1.17 mM of MC3 or 7.1×10 17 MC3 molecules/mL. Considering the LNP concentration (1.3×10 13 LNP/mL) we obtain an average number of ~55000 MC3 molecules/particle.   Figure S4. Identifying the role of MC3 cationic ionizable lipid in LNPs electrostatic adsorption by binding them at pH 4.6 followed by stepwise pH increment. 12%, 35% and 8% release of bound LNPs from the surface was observed for subsequent increasing of the pH from 4,6 to 5.6, 6.6, and 7.6. Majority of desorption happened at the increment from pH 5.6 to 6.6 (64% of the total desorption). This range coincides with the apparent pKa of MC3 LNPs that is reported to be 6.4, 1

Protonation of MC3 lipid and the pH-sensitive interaction of LNPs with ncSLB
which is also what was determined in this study (see Main text). presents the concentration level. Areas without color has concentration below detection limit.

Lipoprotein depleted serum preparation and validation
LFQ: label-free quantification. Figure S6. Typical temporal behavior of LNP local intensities on the surface, over time, following the acidification of environment and pH-induced electrostatic bindings. While kinetics with one step signal increase were abundant, two step increments were rarely observed. Note that the stepwise increase of the intensity is typically in the range 1.1 to 2.5 (e.g., panels A to G) and rarely appreciably larger (e.g., panels H to J). We associate the former stepwise events with LNP collapse whereas the latter events with LNP aggregation.

Characterization of lipid translocation to an ncSLB upon pH-induced LNP collapse
To investigate the nature of the pH-induced interaction between LNPs and a negatively charged SLB in further detail, LNPs containing 0.06 mole% rhodamine labeled DOPE lipids were prepared as described in the Main text, but with slightly altered lipid composition (see the caption of Figure   S7) to obtain LNPs with a mean diameter of ~130 nm, 3

TIRF intensity of attached LNPs
The TIRF intensity of an attached LNP depends on its size and shape and the decay length, δ, of the intensity (square of the amplitude) of the exponentially vanishing evanescent field and can be represented as , where J0 is the intensity of the incident light, vc is the core volume (i.e., a part of the LNP volume containing dye), cd is the dye concentration in the core, ≤ 1 is the factor taking the evanescent field into account, and A is the sensitivity factor. The LNP size (diameter) in the intact state and δ are comparable, and accordingly before collapse the reduction of the TIRF intensity due to the extinction of the evanescent field, i.e., since < 1, is not negligible provided the LNP deformation is modest. Upon LNP collapse, it is reasonable to consider that the dye molecules are integrated with the SLB, and accordingly is close to unity. Thus, the increase of the TIRF intensity upon collapse is equal 1/ , where is the factor corresponding to the LNP state before collapse. The corresponding expression for was obtained in ref. 5 (Eq. 10 there) in the context of localized surface plasmin resonance in the case when a spherical particle directly contacts the sensor surface.
In that case, is a function of the ratio of the particle radius, r, and δ, i.e., = F (r/δ) (Eqs.

Interaction between LNPs in solution
To understand whether LNPs can aggregate in biofluids containing primarily water, it is instructive to estimate the interaction between them. It can be done in the framework of the conventional DLVO theory representing this interaction as a sum of the van der Waals (vdW), hydration, and double-layer electrostatic parts, UvdW, Uh, and Udl. 6 The former two forces operate on the length scale of ≃ 1 nm, while the range of the latter forces depends on the NP size and is appreciably larger. In general, all these forces should be considered. Bearing in mind the physiological conditions, one can, however, notice that the double-layer interaction between identical LNPs is usually rather weak and repulsive (provided the associated charges are of the same sign) and does not result in aggregation. In addition, the parameters available to calculate this interaction are not accurate. For these reasons, we exclude Udl from our analysis and represent the interaction between two LNPs as . ( To calculate UvdW, we use the conventional additive Hamaker approximation (see ref. 7 and references therein), where ALNP-LNP is the Hamaker constant, d is the minimal NP-NP distance, and , , ≡ ln .
The corresponding hydration energy is given by 7 The LNP-LNP interaction, calculated with these parameters as a function of d for the LNP sizes of interest or, more specifically, for the average radius, 40 nm, and maximum radius, 60 nm, is relatively weak ( Figure S9) and cannot result in aggregation of LNPs in solution.

Specifics of measurements under flow conditions
Our experiments were performed under flow conditions. In adsorption experiments with biological molecules (e.g. proteins) or nanoparticles (e.g. vesicles or LNP as in our case), such conditions are preferable because the attachment of such species is often globally controlled by diffusion, and the analysis of the corresponding diffusion-limited adsorption kinetics under the flow conditions is simpler than that under the stagnant (no macroscopic flow) conditions.
Regarding the flow conditions, one can wonder whether the flow influences the rate of kinetic processes occurring locally near the interfaces. This effect is usually axiomatically assumed to be negligible. To validate this assumption in our cases, we can notice that the scale of the solution velocity near the interface or, more specifically, around an attached LNP is given by where is the maximum flow rate, is the scale of the channel size in the direction perpendicular to the flow, and is the LNP radius. According to the Stokes model, 9 the scale of the viscosityrelated force acting on a LNP is where is the coefficient of dynamic viscosity. Substituting (5) into (6) yields ≅ 6 / .
Using Eq. (9) with = 1×10 -3 Ns/m 2 , = 1.6×10 -3 m/s, = 400 m, and = 40 nm (see Materials and Methods for volumetric flow rate and channel dimensions), we obtain < 1 fN. For comparison, one can notice that the forces corresponding to attachment or reconfiguration of LNPs are usually much larger than around 10 / = 20 pN ( ≅ 2 nm is the corresponding length scale). The latter force is much larger than that estimated above by using Eq. (9), and accordingly the effect of the solution flow on the processes under consideration is negligible.